The Problem: Gender differences exist in adaptive and innate immunity, but remain poorly understood. One example is the common and serious problem of pneumonia, where females show greater resistance than males. Although estrogen mediates numerous effects on the immune system, little progress has been made in identifying the specific cellular and molecular mechanisms by which estrogen improves lung host defense. Pilot Data: Using a mouse model of the most common form of pneumonia, pneumococcal pneumonia, we observe greater resistance to infection in females or estrogen-treated males, demonstrated by better clearance of bacteria in vivo, diminished lung inflammation and better survival. We also identify a critical role for nitric oxide generated by constitutively expressed nitric oxide synthase-3 (aka endothelial NOS or NOS3), most likely after priming for NO release by estrogen-mediated signaling events. Hypothesis: Estrogen- mediated activation of NOS3 via PI3K-Akt phosphorylation enhances bacterial killing by AMs and is the basis for greater resistance to pneumonia in females. Specific Aims: Aim 1 will test the postulate that NOS3-production of NO acts within phagosomes where it interacts with NADPH oxidase-derived superoxide to form the potent microbicide, peroxynitrite. In vivo studies will measure clearance, pneumonia and survival in normal mice or mice genetically deficient in NADPH oxidase, endothelial or inducible nitric oxide synthase (NOS3, NOS 2). In vitro analysis of AMs from these mice will measure phagocytosis and killing. Aim 2 will test the postulate that estrogen augments AM killing of pneumococci by activating NOS3 through a PI3K-Akt phosphorylation pathway. Estrogen effects on phosphorylation status and function of NOS3 and Akt will be tested using Western blot analysis, pharmacologic inhibitors, and knockout (KO) mice. The role of a phagocytosis-triggered calcium flux in activating estrogen-primed NOS3 will be evaluated by live microscopy. Aim 3 will test the prediction that host defense against pneumonia will decline in mouse models of menopause, and that estrogen hormone replacement therapy (HRT) will reverse this decline and restore the normal increased female resistance to pneumococcal pneumonia. Significance: This research will identify novel mechanisms in host defense against pneumonia and will inform public health policy about a potential benefit of hormone replacement therapy.